Clinical material collection and culture technique
Collection of mucus in the clinic
The technique of mucus sampling described here is published by Ponikau et al (1999) Two puffs of phenylephrine hydrochloride 1% are sprayed into each nostril, for vasoconstriction to increase the nasal lumen and, consequently, the yield. After two minutes, the patient is asked to breathe inward deeply and to hold the inspiration to occlude the nasopharynx. Each side of the nose is separately flushed with 10ml of sterile 0.9% saline. The equipment used (curved suction device e.g. von Eicken antrum cannula by Karl Storz and 20ml single-use syringe) has to be sterile. The patient is then asked to forcefully exhale through the nose with maximal pressure directly into a sterile 50ml Falcon tube. The material obtained is then submitted to the laboratory within 1 to 2 hours (keep cool).
Sample preparation (Braun et al, 2003; Buzina et al, 2003).
To prevent contamination with omnipresent airborne fungi, all preparation and incubation steps, should be handled under a laminar flow box or hood. To release the fungal elements from the viscous mucus and allow them to contact the growth medium, it is necessary to break apart the disulfide bonds of the mucus. Therefore, each specimen is suspended with an equal volume of diluted dithiothreitol (1.055mg/ml) (or 0.05 parts dithiothreitol) (Sputolysin, Calbiochem, San Diego, CA). The specimen is then vortexed and incubated for 15 minutes at room temperature, followed by a 10 minutes centrifugation at 3000rpm. The supernatant is discarded and the sediment homogenized by vortexing. The sediment is then inoculated on Sabouraud glucose agar plates and malt extract agar plates. Additional media that may be useful include Czapek-Dox or brain heart infusion media. Media should contain antibiotics e.g. chloramphenicol 100mg/l and gentamicin 40mg/l to prevent bacterial overgrowth. These plates are then incubated at 30oC for up to 6 weeks. All cultures are examined at 3-day intervals and each isolate identified. Isolates failing to sporulate can be subcultured into corn meal or potato dextrose agar.
Collection of Surgical Specimens and Histological Examination.
The principle of maximum mucus preservation is required to optimise yield during the acquisition of surgical specimens. To ensure maximal mucous collection, all surgical procedures should be performed without a power microdebrider or the use of suction devices until sample collection is complete. Mucus should be manually removed, together with inflamed tissue, and placed on a saline-moistened sheet of sterile used x-ray film (approx. 10 x 10 cm) to prevent absorption of the mucus. It should not be placed on a surgical towel or gauze. Each specimen is then fixed in 10% formalin and embedded in paraffin as for other histology specimens. Multiple serial sections of different specimens from each patient were stained with H & E and with Gomori methanamine silver. The pathologists were alerted to pay special attention to the mucin, focusing on fungal elements and eosinophils.
Buzina W, Braun H, Freudenschuss K, Lackner A, Habermann W, Stammberger H. Fungal biodiversity-as found in nasal mucus. Med Mycol 2003;41:149-61.
Braun H, Buzina W, Freudenschuss K, Beham A, Stammberger H. Eosinophilic fungal rhinosinusitis: a common disorder in Europe? Laryngoscope. 2003;113:264-9.